A continuous cell culture method based on a cultivation hybrid model

Abstract

The present invention relates to the technical field of cell culture, and specifically discloses a continuous cell culture method based on a culture mixing model, including: obtaining cell culture environment data; segmenting the environment data according to a preset culture time, and calculating the statistical features and analysis features of the environment data in each culture time period; performing factor analysis on the statistical features to obtain culture features; performing integrated analysis on the analysis features to obtain mixing features; classifying the culture features into multiple culture types, and establishing recognition rules based on the culture features of the category center; using database analysis to classify the mixing features into multiple mixing types, and establishing judgment rules based on the mixing types of professional judgment; combining the recognition rules and the judgment rules to establish a composite discrimination rule; collecting and inputting current cell culture data, and outputting a cell culture method based on the composite discrimination rule. Through this culture method, the mixing state of cell culture can be efficiently adjusted, thereby improving the success rate of continuous cell culture.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular, to a continuous cell culture method based on a culture mixing model. Background Art

[0002] Continuous culture refers to a culture method in which a microbial strain is inoculated in a non-closed culture system, and fresh nutrient solution is continuously supplemented during the culture process to remove inhibitory factors, optimize the growth environment, and continuously collect the culture product. Since continuous culture is formulated and implemented based on in-depth research on the growth curve of microorganisms in batch culture, it has significant characteristics and advantages. It can, to a certain extent, artificially control a certain period in the growth curve according to the purpose of the researcher, making it shorter or longer, or accelerating / decelerating the cell growth rate in a certain period, thereby greatly improving the controllability of the microbial culture process.

[0003] Cell culture refers to a technique in which cells grow, reproduce, and maintain their main structures and functions under artificially created conditions similar to the in-vivo environment, such as appropriate temperature, pH, and certain nutrients. For example, the Chinese invention patent with the publication number CN113286650A discloses a continuous cell culture method for continuously culturing shear-sensitive cells in a bioreactor system with a controlled gas outlet velocity. Specifically, a cell population composed of shear-sensitive cells is cultured in a bioreactor system with at least 25 L of culture medium and a gas outlet velocity of at most 20 m / s to achieve a steady-state viable cell concentration in the range of 20×106 cells / mL to 15×107 cells / mL in the culture medium. According to the type of cells, cell culture is divided into primary culture and subculture, and according to the growth characteristics of cells, it can be further divided into adherent culture and suspension culture. Primary culture refers to the process of directly separating cells from tissues and culturing them in cell culture flasks or culture dishes. When cells grow and reproduce continuously, they are closely attached to each other, with a small space, high density, insufficient nutrients, and accumulation of metabolites, resulting in a slow cell growth rate, poor state, and even death. Therefore, subculture is required to avoid this situation. Subculture is the process of digesting and diluting cells and aliquoting them into two or more culture flasks or culture dishes for continued growth. Adherent culture refers to the process in which adherent-dependent cells attach to a certain solid phase surface for growth and reproduction. Suspension culture refers to the process in which some cells that do not have the characteristics of adherent growth can only grow and reproduce in a flowing liquid culture medium. Among them, during suspension culture, the culture solution needs to be shaken.

[0004] Therefore, during the continuous suspension culture of cells, continuous shaking culture of the cells is required. Shaking culture refers to a culture method in which microbial cells are inoculated into a liquid medium and placed on a shaker or oscillator for continuous shaking. For example, the Chinese invention patent with the publication number CN104759226A discloses a shaker and a cell suspension culture method that can be used in a carbon dioxide incubator. The shaker includes a power supply system, a driving device, and an execution mechanism. The power supply system and the driving device are connected by a power cord that can cross the inside and outside of the incubator through the mating part of the inner door and the gasket of the door frame of the incubator or through the through hole on the back of the incubator. The power cord can separate the driving device and the execution mechanism from the power supply system inside and outside the incubator, thus solving the problems of overheating caused by heat accumulation in the power supply system when an ordinary shaker is used in a carbon dioxide incubator and damage to its electronic components due to moisture.

[0005] Oscillation can make the medium fully contact with oxygen, increase the supply of dissolved oxygen, and the bacteria cultured by shaking reproduce uniformly with high culture efficiency. In the process of continuous cell culture, the oscillation time is relatively long, and oscillation has a great impact on the results of continuous cell culture. However, in actual use, due to the single frequency of the oscillation device and the relatively simple setting of the oscillation amplitude, the oscillation effects of the upper-layer cells and the lower-layer cells are very different during the continuous cell culture process, and the contact degrees of the cells in different parts of the medium with the culture solution and with oxygen also vary greatly, resulting in a low success rate of continuous cell culture. Summary of the Invention

[0006] The present invention provides a continuous cell culture method based on a culture mixing model, which can efficiently adjust the mixing state of cell culture through this culture method, thereby improving the success rate of continuous cell culture.

[0007] The technical solution adopted by the present invention to solve the above technical problems is as follows: A continuous cell culture method based on a culture mixing model includes the following steps:

[0008] S1: Obtain cell culture environment data, where the environment data includes culture solution state data and mixer state data. The culture solution state data includes culture solution temperature, culture solution pH value, and culture solution oxygen content. The mixer state data includes mixing method, mixing frequency, and mixer oxygen content;

[0009] S2: Segment the environment data according to a preset culture time, and calculate the statistical characteristics and analysis characteristics of the environment data within each culture time period;

[0010] S3: Perform factor analysis on the statistical characteristics to obtain culture characteristics; at the same time, perform integrated analysis on the analysis characteristics to obtain mixing characteristics;

[0011] S4: Classify the culture characteristics into multiple culture types, and establish recognition rules based on the culture characteristics of the category center;

[0012] S5: Use database analysis to classify the mixed characteristics into multiple mixed types, and establish judgment rules based on the mixed types of professional judgment;

[0013] S6: Combine the recognition rules and the judgment rules to establish a composite discrimination rule;

[0014] S7: Collect and input the current cell culture data, and output the cell culture method based on the composite discrimination rule.

[0015] Furthermore, step S2 specifically includes:

[0016] S211: Segment the environmental data at time intervals of T2 minutes to obtain multiple second culture segments;

[0017] S212: Calculate the mixing mode coefficient θ, the average mixing frequency L1, and the average oxygen content Q1 of the mixer in each second culture segment as analysis characteristics;

[0018] S221: Segment the environmental data at time intervals of T1 minutes to obtain multiple first culture segments;

[0019] S222: Calculate the average culture solution temperature C1, the standard deviation P1 of the pH value of the culture solution, and the average oxygen content H1 of the culture solution in each first culture segment as statistical characteristics;

[0020] Among them, the mixing mode coefficient θ is determined by the mixing mode used in the second culture segment.

[0021] Furthermore, the following relationship is satisfied between T1 and T2:

[0022]

[0023] Among them, μ represents the mixing frequency coefficient, γ represents the oxygen-containing risk coefficient, the mixing frequency coefficient is determined by the deviation degree between the average mixing frequency L1 and the set mixing frequency L s and the oxygen-containing risk coefficient is determined by the deviation degree between the average oxygen content Q1 of the mixer and the set oxygen content Q of the mixer s between.

[0024] Furthermore, the culture characteristics include the first culture characteristic X1 and the second culture characteristic X2, and the first culture characteristic X1 and the second culture characteristic X2 are respectively obtained through the following methods:

[0025]

[0026]

[0027] Among them, C0 represents the set optimal culture medium temperature, P0 represents the maximum set difference value of the pH value of the culture medium, H0 represents the set optimal oxygen content of the culture medium, δ represents the culture medium temperature factor, ρ represents the culture medium oxygen content factor, represents the culture medium temperature factor, ln() represents the logarithmic function with the constant e as the base, max(C1, C0) represents taking the maximum value between C1 and C0, and min(P0, P1) represents taking the minimum value between P0 and P1.

[0028] Furthermore, the analysis features include a first analysis feature Y1 and a second analysis feature Y2, and the first analysis feature Y1 and the second analysis feature Y2 are respectively obtained through the following methods:

[0029]

[0030]

[0031] Among them, L0 represents the set optimal mixing frequency, Q0 represents the set optimal oxygen content of the mixer, α represents the mixing frequency factor, β represents the mixer oxygen content factor, represents the logarithmic function with L0 as the base, represents the logarithmic function with Q0 as the base, and sin() represents the sine function, is the θ power of Y1.

[0032] Furthermore, in step S4, the first culture feature X1 and the second culture feature X2 are classified into N different culture types through a two-dimensional coordinate axis, and the difference in the number of culture features between every two culture types is less than 20; where N ∈ (4, 16).

[0033] Furthermore, in step S4, the culture feature F at the center is selected from each culture type ij , F ij contains and F ij represents the culture feature at the center of the i×jth culture type, and respectively represent the first culture feature value and the second culture feature value at the center of the i×jth culture type, and the following recognition rules are established:

[0034]

[0035]

[0036] Among them, represents the input first culture feature value, Represents the second culture characteristic value of the input.

[0037] Further, in step S5, the first analysis feature Y1 and the second analysis feature Y2 are input into the established database, and the mixed features are classified into M different mixed types according to the value Z output by the database.

[0038] Further, in step S5, one of the M mixed types is selected, and the corresponding value Z output by the database is determined according to the selected mixed type, and the following judgment rule is established:

[0039]

[0040] Wherein, Represents the first analysis feature value of the input, Represents the value of the second analysis feature of the input, q represents the first analysis feature value adjustment factor, and p represents the second analysis feature value adjustment factor.

[0041] Further, in step S6, the judgment rule is used as the first operation rule. After the judgment rule can run, the recognition rule is started to run, thereby establishing a composite discrimination rule.

[0042] Compared with the prior art, the advantages of the present invention are: by analyzing the detection data of the mixing method, mixing frequency, oxygen content of the mixer in the mixer for successful continuous cell culture and the detection data of the culture solution temperature, culture solution pH value, and culture solution oxygen content at the same time, a feasible culture mixing model is established, thereby improving the replicability and success rate of continuous cell culture; moreover, multiple culture types and mixing types are established in the method of the present application, increasing the mixing method and frequency and the culture method, which can effectively improve the quality of continuous cell culture, and at the same time reduce the situation of relying on manual recognition and manual adjustment during the culture process, and reduce the labor cost of the culture. Specific Embodiments

[0043] In order to make the above objects, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below.

[0044] This embodiment provides a continuous cell culture method based on a culture mixing model, and the specific steps are as follows:

[0045] S1: Obtain cell culture environment data, the environment data includes culture solution state data and mixer state data, the culture solution state data includes culture solution temperature, culture solution pH value, culture solution oxygen content, and the mixer state data includes mixing method, mixing frequency, mixer oxygen content.

[0046] The obtained cell culture environment data is mainly detected from tests with successful continuous cell culture, and the obtained cell culture environment data includes the environment data of at least 5 groups of the same type of cells. The method of this application mainly detects the state of the culture medium and the state of the mixer during the continuous cell culture process, so as to obtain the data of the overall culture environment during the continuous cell culture process.

[0047] S2: Segment the environment data according to the preset culture time, and calculate the statistical features and analysis features of the environment data in each culture time period;

[0048] Segment the environment data at intervals of T2 minutes to obtain multiple second culture segments; calculate the mixing mode coefficient θ, the average mixing frequency L1, and the average oxygen content Q1 of the mixer in each second culture segment as analysis features; segment the environment data at intervals of T1 minutes to obtain multiple first culture segments; calculate the average culture medium temperature C1, the standard deviation P1 of the pH value of the culture medium, and the average oxygen content H1 of the culture medium in each first culture segment as statistical features.

[0049] The following relationship is satisfied between T1 and T2:

[0050]

[0051] where μ represents the mixing frequency coefficient, γ represents the oxygen-containing risk coefficient, the mixing frequency coefficient is determined by the deviation between the average mixing frequency L1 and the set mixing frequency L s and the oxygen-containing risk coefficient is determined by the deviation between the average oxygen content Q1 of the mixer and the set oxygen content Q of the mixer s and the mixing mode coefficient θ is determined by the mixing mode used in the second culture segment.

[0052] In step S2, first segment the obtained data according to time, and subsequent analysis can be carried out according to time periods. Among them, the obtained culture medium state data is segmented at intervals of T2 minutes, T2 ∈ [5, 10], and the obtained mixer state data is segmented at intervals of T1 minutes. The above relationship is satisfied between T1 and T2. Limiting the relationship between T1 and T2 can obtain a more reasonable segmentation of the mixer state data. Through the comprehensive judgment of the average mixing frequency L1, the average oxygen content Q1 of the mixer, and the mixing mode coefficient θ, the relationship between the mixing mode and the mixing frequency can be balanced, and at the same time, the oxygen content situation in the mixer improves the division of T1 as a whole.

[0053] In step S2, the analysis features are defined as the mixing mode coefficient θ, the average mixing frequency L1, and the average oxygen content Q1 of the mixer within each second culture segment. The mixing modes mainly include two types: stirring and oscillation. The stirring type includes central swirling and axial stirring, and the oscillation type mainly includes horizontal oscillation, rocking oscillation, amplitude-varying oscillation, undulating oscillation, up-and-down fluctuation oscillation, and three-dimensional space oscillation. Different mixing mode coefficients θ are assigned according to different mixing modes. When taking the average of the mixing frequency and the oxygen content of the mixer, it is also necessary to denoise the data and remove unreasonable values.

[0054] In step S2, the statistical features are also defined as the average culture solution temperature C1, the standard deviation P1 of the pH value of the culture solution, and the average oxygen content H1 of the culture solution within each first culture segment. Since the change rates of the culture solution temperature and the oxygen content of the culture solution are relatively slow, the average values can be used to represent them. When taking the average of the culture solution temperature and the oxygen content of the culture solution, it is also necessary to denoise the data and remove unreasonable values. Since the change rate of the pH value of the culture solution is relatively fast and relatively sensitive, it is represented in the form of the standard deviation.

[0055] S3: Perform factor analysis on the statistical features to obtain culture features; the culture features include the first culture feature X1 and the second culture feature X2, and the first culture feature X1 and the second culture feature X2 are obtained through the following methods respectively:

[0056]

[0057]

[0058] Among them, C0 represents the set optimal culture solution temperature, P0 represents the maximum set difference of the pH value of the culture solution, H0 represents the set optimal oxygen content of the culture solution, δ represents the culture solution temperature factor, and ρ represents the culture solution oxygen content factor. represents the culture solution temperature factor, ln() represents the logarithmic function with the constant e as the base, max(C1, C0) represents taking the maximum value between C1 and C0, and min(P0, P1) represents taking the minimum value between P0 and P1.

[0059] At the same time, perform integrated analysis on the analysis features to obtain mixing features; the analysis features include the first analysis feature Y1 and the second analysis feature Y2, and the first analysis feature Y1 and the second analysis feature Y2 are obtained through the following methods respectively:

[0060]

[0061]

[0062] Among them, L0 represents the set optimal mixing frequency, Q0 represents the set optimal oxygen content in the mixer, α represents the mixing frequency factor, and β represents the oxygen content factor of the mixer. represents the logarithmic function with L0 as the base. represents the logarithmic function with Q0 as the base, and sin() represents the sine function. is the θ power of Y1.

[0063] In step S3, the cultivation characteristics are represented by the first cultivation characteristic X1 and the second cultivation characteristic X2. The first cultivation characteristic X1 is used to reflect the temperature of the culture solution, and the second cultivation characteristic X2 is used to reflect the overall situation of the culture solution. In step S3, the mixing characteristics are also represented by the first analysis characteristic Y1 and the second analysis characteristic Y2. The first analysis characteristic Y1 is used to reflect the comprehensive situation of the mixing frequency and the oxygen content in the mixer, and the second analysis characteristic Y2 is used to represent the overall situation of the mixer.

[0064] S4: Classify the cultivation characteristics into multiple cultivation types, and establish recognition rules based on the cultivation characteristics at the category center: Classify the first cultivation characteristic X1 and the second cultivation characteristic X2 into N different cultivation types through a two-dimensional coordinate axis, and the difference in the number of cultivation characteristics between every two cultivation types is less than 20; where N ∈ (4, 16), and select the cultivation characteristic F at the center from each cultivation type. ij F ij contains and F ij represents the cultivation characteristic at the center of the i×j-th cultivation type. and represent the first cultivation characteristic value and the second cultivation characteristic value at the center of the i×j-th cultivation type respectively, and establish the following recognition rules:

[0065]

[0066]

[0067] Among them, represents the input first cultivation characteristic value. represents the input second cultivation characteristic value.

[0068] Step S4 is to obtain the aggregated cultivation types through the classification of cultivation characteristics, so as to facilitate the rapid adjustment of the cultivation methods in the subsequent steps.

[0069] S5: Classify the mixed features into multiple mixed types using database analysis, and establish judgment rules based on the mixed types judged by professionals: Input the first analysis feature Y1 and the second analysis feature Y2 into the established database. Classify the mixed features into M different mixed types through the value Z output by the database. Select one of the M mixed types, and determine the corresponding value Z output by the database according to the selected mixed type, and establish the following judgment rules:

[0070]

[0071] Among them, represents the input value of the first analysis feature, represents the value of the second analysis feature input, q represents the adjustment factor of the first analysis feature value, and p represents the adjustment factor of the second analysis feature value.

[0072] In step S5, establish a database using the currently known cell culture mixing methods, and assign values according to the effects of the mixing methods in the database, that is, the value Z output by the database. Then classify the mixed features into M different mixed types according to the value Z output by the database.

[0073] S6: Establish an operating order for the judgment rules and recognition rules: Use the judgment rules as the first operating rule. After the judgment rules can operate, start running the recognition rules to establish composite discrimination rules.

[0074] S7: Collect and input the current cell culture data, and output the cell culture method based on the composite discrimination rules.

[0075] In step S7, according to the established operation rules, input the currently collected cell culture data. First, use the judgment rules to judge whether the current mixer state conforms to the rules. After the mixer state conforms to the operating conditions, start judging the state of the culture medium, and finally output the discriminated cell culture method.

[0076] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be determined by the scope defined by the claims.

Claims

1. A continuous cell culture method based on a cultivation hybrid model, characterized in that, It includes the following steps: S1: Obtain cell culture environment data, where the environment data includes culture solution state data and mixer state data. The culture solution state data includes culture solution temperature, culture solution pH value, and dissolved oxygen content in the culture solution. The mixer state data includes mixing method, mixing frequency, and dissolved oxygen content in the mixer; S2: Segment the environment data according to a preset culture time, and calculate the statistical features and analysis features of the environment data in each culture time period; S3: Conduct factor analysis on the statistical features to obtain culture features; at the same time, conduct integrated analysis on the analysis features to obtain mixing features; S4: Classify the culture features into multiple culture types, and establish recognition rules based on the culture features at the category center; S5: Use database analysis to classify the mixing features into multiple mixing types, and establish judgment rules based on the mixing types judged by professionals; S6: Combine the recognition rules and the judgment rules to establish a composite discrimination rule; S7: Collect and input current cell culture data, and output the cell culture method based on the composite discrimination rule; The specific content of step S2 includes: S211: Segment the environmental data at intervals of minutes to obtain multiple second culture segments; S212: Calculate the mixing mode coefficient θ, average mixing frequency , and average mixer oxygen content within each second culture segment as the analysis feature; S221: Segment the environmental data at intervals of minutes to obtain multiple first culture segments; S222: Calculate the average culture solution temperature within each first culture segment , the standard deviation of the pH value of the culture solution , the average dissolved oxygen content of the culture solution , as the statistical feature; Among them, the mixing method coefficient θ is determined by the mixing method used in the second culture segment; The cultivation characteristics include the first cultivation characteristic and the second cultivation characteristic The first cultivation characteristic and the second cultivation characteristic are respectively obtained through the following methods: ； ； Among them, represents the set optimal culture medium temperature, represents the maximum set difference value of the culture medium pH value, represents the set optimal oxygen content of the culture medium, represents the culture medium temperature factor, represents the oxygen content factor of the culture medium, represents the culture medium temperature factor, and ln( ) represents the logarithmic function with the constant e as the base, represents taking the maximum value between, represents taking the minimum value between; The analysis features include a first analysis feature and a second analysis feature . The first analysis feature and the second analysis feature are respectively obtained through the following methods: ； ； Among them, represents the set optimal mixing frequency, represents the set optimal oxygen content in the mixer, represents the mixing frequency factor, represents the oxygen content factor in the mixer, ( ) represents the logarithmic function with as the base, () represents the logarithmic function with as the base, and sin( ) represents the sine function, is to the power; in the step S4, select the cultivation characteristics at the center from each of the said cultivation types , contains and , , , represents the cultivation characteristics at the center of the th cultivation type, and respectively represent the first cultivation characteristic value and the second cultivation characteristic value at the center of the th cultivation type, and establish the following recognition rules: ； ； Among them, represents the first culture eigenvalue of the input, represents the second culture eigenvalue of the input; In step S5, the first analysis feature And the second analysis feature Input into the established database, and classify the mixing characteristics into M different mixing types according to the value Z output by the database; In step S5, select one of the M mixing types, and determine the corresponding value Z output by the database according to the selected mixing type, and establish the following judgment rule: ; Among them, represents the first analysis eigenvalue of the input, represents the value of the second analysis feature of the input, represents the first analysis eigenvalue adjustment factor, represents the second analysis eigenvalue adjustment factor.

2. The continuous cell culture method according to claim 1, wherein Said With the The following relationship is satisfied: ； Among them, represents the mixing frequency coefficient, represents the oxygen-containing risk coefficient, and the mixing frequency coefficient is determined by the deviation between the average mixing frequency and the set mixing frequency , and the oxygen-containing risk coefficient is determined by the deviation between the average oxygen content of the mixer and the set oxygen content of the mixer .

3. The continuous cell culture method according to claim 1, wherein In the step S4, the first culture feature is classified by a two-dimensional coordinate axis and the second culture feature into N different culture types, and the difference in the number of the culture features between every two culture types is less than 20; where N ∈ (4, 16).

4. The continuous cell culture method according to claim 1, wherein In step S6, take the judgment rule as the first operation rule. After the judgment rule can run, start running the recognition rule, so as to establish the composite discrimination rule.

Images